I have 2 cDNA samples to which I have added two different adaptors, adator 1 to 1 cDNA and adator 2 to another cDNA. When I combined the 2 samples of single strand cdna to get double strand cDNA, I encounter the adaptor part overhangs on 5' and 3'. How do I fill the gap and how to perform the reaction. Please give your valuable suggestion in this regard.
Hi,
filling-in 5´ overhangs is very easy, just combine your sample with a mixture of dNTPs and synthetize it with eg Klenow Fragment or T4 DNA polymerase.
Filling in 3ˇoverhangs is a bit more complicated. If you need just to create blunt end, you can use T4 DNA polymerase - but it will not fill in, but rather cleave off the overhang (you will loose the overhang).
Should you need to preserve overhanging sequence, you would need to PCR amplify your sample with a primer, that contains your overhang.
Hi,
filling-in 5´ overhangs is very easy, just combine your sample with a mixture of dNTPs and synthetize it with eg Klenow Fragment or T4 DNA polymerase.
Filling in 3ˇoverhangs is a bit more complicated. If you need just to create blunt end, you can use T4 DNA polymerase - but it will not fill in, but rather cleave off the overhang (you will loose the overhang).
Should you need to preserve overhanging sequence, you would need to PCR amplify your sample with a primer, that contains your overhang.
Thank you very much....Could you please tell me at what temperature and for how much time I have to run PCR so that the 5' overhangs fill...utilizing T4 DNA polymerase with dNTP. Could your briefly write the reaction and the contents utilize for the reaction....
Do it according your supplier recommendation. I am using polymerase from Fermentas (https://www.lifetechnologies.com/order/catalog/product/EP0061?ICID=search-product), but you can try NEB (https://www.neb.com/protocols/2014/01/13/protocol-for-blunting-ends-by-3-overhang-removal-and-fill-in-of-3-recessed-5-overhang-ends-using1) or other.
Generally, do not allow the reaction to proceed for a long time or at high temperature - it might cleave off more than desired. Typical time is 5 min at room temp to approx 20 min at 11 or 12°C. Heat inactivate enzyme and you have blunt ended product (filled in 5´overhang, removed 3´overhang).
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